One focus of the group is to determine how Escherichia coli and Saccharomyces cerevisiae cells sense and defend against oxidative stress. Reactive oxygen species can lead to the damage of almost all cell components (DNA, lipid membranes, and proteins) and have been implicated as causative agents in several degenerative diseases. However, most organisms can induce defenses against these oxidants, and it is our goal to understand these adaptive responses. In bacterial cells, a key regulator of the response to hydrogen peroxide is the OxyR transcription factor. OxyR is both the sensor and transducer of the oxidative stress signal; the oxidized but not the reduced form of the purified regulator can activate transcription in vitro. The group previously found that OxyR is activated by the formation of an intramolecular disulfide bond between C199 and C208 and is deactivated by enzymatic reduction by glutaredoxin 1 together with glutathione. Recent structural studies showed that formation of the C199-C208 disulfide bond leads to a large conformational change. In additional experiments, OxyR binding sites in the E. coli genome were identified using a computational approach, and the transcription profile of the E. coli response to hydrogen peroxide was determined by microarrays. The chemical basis of OxyR sensitivity to hydrogen peroxide and the roles of all of OxyR target genes now are being investigated. Compared to the bacterial responses to hydrogen peroxide, little is known about the induction of eukaryotic defenses against oxidative stress. Thus, the group carried out microarray experiments to determine genomic expression programs induced by hydrogen peroxide in wild type and mutant S. cerevisiae strains. These studies confirmed that the Yap1 transcription factor is critical for the hydrogen peroxide-dependent induction of many genes. Genetic screens to isolate mutations in components of the yeast signal transduction pathways identified thioredoxin reductase as playing a role in modulating Yap1 activity. Currently, the group is isolating and characterizing additional mutants and analyzing the purified Yap1 protein. A second focus of the group is to elucidate the functions of small, untranslated RNAs. More and more of these RNAs have been shown to play important regulatory roles. One of the OxyR-induced genes encodes the OxyS RNA that acts as a pleiotropic regulator and as an antimutator. OxyS RNA action requires the Hfq protein, and biochemical experiments showed that Hfq binds to the OxyS RNA. This past year, the group found that Hfq is a bacterial homolog of Sm and Sm-like proteins integral to RNA processing and mRNA degradation in eukaryotic cells. The nature of the OxyS RNA-Hfq protein interaction now is being characterized. Programs used to identify protein-encoding genes generally do not detect small RNA-encoding genes. To try to identify more of the small RNAs encoded by the E. coli genome, the group utilized comparative genomics and microarrays. These approaches led to the identification of 17 new small RNAs, many of which bind to the Hfq protein. Experiments to examine the global role of Hfq and to elucidate the functions of the newly identified small RNAs are underway.